Method

ABSTRACT

The method of the invention relates to a method of typing one or more nucleic acid molecules, said method comprising: simultaneously or sequentially performing two or more primer extension reactions, each primer binding at a different predetermined site in said nucleic acid molecule(s), and determining the pattern of nucleotide incorporation to obtain a test pattern for said nucleic acid molecule(s) which is optionally compared with one or more reference patterns to type the said nucleic acid molecule(s).

This invention relates to a method of typing using nucleic acid, and inparticular to an improved method of genotyping.

Typing, e.g. genotyping, can be particularly advantageous for medicaldiagnosis, prognosis and treatment. For example, identification of themicrobe responsible for infection allows correct treatment to beadministered. It has been shown that microbes may now readily beidentified by typing (i.e. by identifying genomic signature patternscharacteristic of a particular microbe). Typing one or more variableregions in a gene or genes or other nucleic acid sequence of anindividual can reveal markers of predisposition to a particular disease,condition or syndrome, and may also point to the best method oftreatment of the foregoing. Typing methods are also useful for genomicanalyses (e.g. in typing polymorphisms or allelic variations), tissuetyping or environmental monitoring and contamination testing etc.

Conventional assays for detection of bacterial or viral species, or fordetecting mutations or polymorphisms in a DNA sequence include using thepolymerase chain reaction (PCR) method. This method is designed topermit selective amplification of a particular target DNA sequence orsequences, determined by the nature of the amplification primers used.To permit such selective amplification, some prior knowledge of thesequence of the DNA is required, enabling the construction of twooligonucleotide primer sequences, known as amplimers. One amplimerhybridises at or towards the 5′ end of one of the strands of the targetDNA and the other amplimer at or towards the 5′ end of the secondstrand. In the presence of a DNA polymerase and DNA precursors (i.e.DATP, dCTP, dGTP and dTTP) the primers can initiate the synthesis of newDNA strands which are complementary to the individual strands of thetarget DNA segment. The use of a heat-stable polymerase enables theprocedure to be readily repeated or cycled. The newly synthesized DNAstrands act as templates for further DNA synthesis in subsequent cycles.The reaction mixture is subjected to a temperature of about 90° C. toseparate the double stranded DNA formed by the polymerase. The reactiontemperature is reduced to about 50° C. to 70° to allow the singlestranded DNA to anneal to the primers, and another round of DNAsynthesis is performed. The DNA synthesized extends between the terminiof the two primers. Preferably, the DNA polymerase used is thermophilici.e. Taq polymerase. After 30 cycles of DNA synthesis, the products ofPCR will include about 10⁵ copies of the specific target sequence. Atypical PCR reaction cycle is therefore: synthesis of the separatestrands by primer extension, separation of strands, primer annealing,synthesis of new strands.

The chain reaction can therefore be perpetuated merely by raising andlowering the temperature.

Typing (e.g. genotyping) be performed using PCR-based techniques, forexample using allele-specific primers (Okamoto et al. 1992, Journal ofGeneral Virology, 73, 673-679; Widell, A et al. 1994, Journal of MedicalVirology, 44, 272-279).

Currently, multiplex PCR may be used to screen samples of nucleic acidsfor a given panel of mutations/variations within the nucleic acidsequence. This method is still cumbersome for routine diagnostics, asthe use of gel electrophoresis is essential. Alternative methods rely onthe use of labelled nucleotides or primers, and may require complexdetection strategies or mechanisms. There is thus a need for a typingmethod which may analyse nucleic acids with respect to 2 or morevariable regions or positions typically without the need for gelelectrophoresis and preferably without the use of labelled nucleotidesor primers.

Other-methods for typing include serologically-based detection methods(Viazov, et al. 1994, Journal of Virological Methods, 48, 81-91;Schroter, M., 1999, Journal of Medical Virology, 57, 230-234), lineprobe assay (Stuyver, L., 1993, Journal of General Virology, 74,1093-1102, Stuyver, L., 1996, Transfusion, 36, 552-558), and restrictionfragment length polymorphism (McOmish et al. 1993, Transfusion, 33,7-13; Buoro, S. 1999, Intervirology, 42, 1-8) are well known in the art.However, sequencing continues to be regarded in the art as the “goldstandard” method for typing. Accordingly, a sequencing-based typingmethod which avoids the drawbacks mentioned above would represent aconsiderable advance in the art.

PCR is also commonly used in the detection of microbes, i.e. bacteria orviruses. However, conventional PCR assays are limited in diagnosticapplications, and generally only indicate whether or not a microbe, or aparticular sequence is present. For many infections, e.g. viralinfections such as hepatitis C viral infection, the infectingmicroorganism may occur in a number of different sub-types, for exampleat least seven subtypes (or genotypes) are known of the HCV virus. Itwould be advantageous in such circumstances not only to determine thatthe general “class” (or genus or species) of infecting microorganism ispresent (e.g. HCV virus), but also to determine which of the sub-typesis present.

Similarly, genomic studies have now revealed that many other diseases ordisorders may be associated with genetic variations (e.g. mutations,allelic variations or polymorphisms (e.g. single nucleotidepolymorphisms, (SNPs)), and that the presence of such variations mayindicate a risk or predisposition to a disease or disorder, or may evenindicate or predict how an individual may respond to a particulartreatment for that disease or disorder (this latter effect is referredto as “pharmacogenomics”). Accordingly, in clinical science, theanalysis (typing) of such variations may be of importance.

Microbial subtypes and clinically informative polymorphisms (or othergenetic variations) frequently are characterised by combinations ofgenetic variations (i.e. variations in multiple (i.e. two or more)positions or regions of the genome etc.). Accordingly, for typingpurposes in such situations it is necessary to “type” (or identify) morethan one variation (polymorphism). In other words, it is necessary totype (or study or identify) a polymorphic pattern (a pattern of geneticvariations) which pattern may cover more than one region of the genometo be studied. (The term “polymorphic pattern” is used herein broadly toinclude patterns, or combinations, of two or more (e.g. 3, 4, 5, 6, 7,8, 9, 10 or more) of any type of genetic variation, e.g. mutations,allelic variants, polymorphisms of any type etc.). It will be understoodthat the variation can be an insertion or deletion of one or morenucleic acid residues.

Thus, for many microorganisms, diseases or predisposition to disease, anidentification of the exact type of microbe, or genetic variation,present is needed to make a proper diagnosis or prognosis and in orderto achieve this it is necessary to study more than one geneticallyvariant position or region. As mentioned above, PCR is an extremelyuseful tool for the amplification and/or identification of a specificsequence of DNA, but to use conventional PCR techniques to determine thegenotype of a nucleic acid molecule based on multiple genetic variationsrequires a repeated and multiple number of individual reactions to beperformed, which would be cumbersome, time-consuming and expensive toperform using conventional technologies and procedures such as e.g.electrophoresis or labelling technologies. There is therefore a need fora typing assay that is accurate and reliable, has a short analysis timeand is quick and easy to perform. The present invention addresses thisneed.

In particular, it has now been found that a simple, reliable, andaccurate method for obtaining typing (sequence) information about aplurality of variable sites within target nucleic acid, may be performedusing a primer extension reaction system using two or more specificprimers designed to bind at or near to these variable sites, allowingprimer extension reactions to be carried out on each primer annealed toa template nucleic acid sequence, either sequentially or simultaneously,and detecting the pattern of nucleotide incorporation in said primerextension reactions. The pattern of nucleotide incorporation providingthe typing information about said variable sites.

This new method of the invention thus combines a multiplexing approach(i.e. an approach relying on the simultaneous or parallel performance ofmultiple reactions) with a particular strategy for detecting the resultof the multiple primer extensions, namely detecting the pattern ofnucleotide incorporation.

The method is particularly suited to automation e.g. in systems wherereaction and reagent dispensing steps take place in a microtitre plateformat. The methods are particularly suitable for identifying microbialspecies and subtypes thereof, but may also find application in othertyping procedures e.g. typing of polymorphisms, e.g. for tissue typingor in clinical applications.

As described further below the present invention is advantageously basedon a method of “sequencing-by-synthesis” (see e.g. U.S. Pat. No.4,863,849 of Melamede). This is a term used in the art to definesequencing methods which rely on the detection of nucleotideincorporation during a primer-directed polymerase extension reaction.The four different nucleotides (i.e. A, G, T or C nucleotides) are addedcyclically or sequentially (conveniently in a known order), and theevent of incorporation can be detected in various ways, directly orindirectly, This detection reveals which nucleotide has beenincorporated, and hence sequencing information; when the nucleotide(base) which forms a pair (according to the normal rules of basepairing, A-T and C-G) with the next base in the template target sequenceis added, it will be incorporated into the growing complementary strand(i.e. the extended primer) by the polymerase, and this incorporationwill trigger a detectable signal, the nature of which depending upon thedetection strategy selected.

Accordingly, the present invention provides a method of typing 1 or morenucleic acid molecules, said method comprising:

-   -   simultaneously or sequentially performing two or more primer        extension reactions, each primer binding at a different        predetermined site in said nucleic acid molecule(s), and        determining the pattern of nucleotide incorporation to obtain a        test pattern (or “fingerprint”) for said nucleic acid molecule        which is optionally compared with one or more reference patterns        to type the said nucleic acid molecule(s).

Preferably, the primer extension reactions occur simultaneously, i.e.both or all primers are annealed and are capable of primer extension atthe same time. It will, of course, be appreciated that each individualprimer can only be extended if a nucleotide is added to the reaction mixwhich is complementary to the next nucleotide in the template. Thus foreach nucleotide addition, not every primer (or even any primer) willactually be extended and the term ‘simultaneous’ must be interpretedwith this in mind.

The method of the invention may be used to type a nucleic acid moleculecontaining two or more sites at which its sequence may be variable(“variable sites”) and each said primer binds at a site lying at or nearto a variable site. Different nucleotides may be added sequentially toperform the primer extension reactions, and are described further below.

Alternatively, the method of the invention may be used to type two ormore nucleic acid molecules containing 1 or more sites at which thesequence may be variable (“variable sites”) and each primer binds at asite lying at or near to a variable site. Different nucleotides may beadded sequentially to perform the primer extension reactions, and aredescribed further below. This embodiment may be particularly useful whenit is desired to obtain information about variable sites within therelated genes, for example SNPs in Factor V Leiden and Prothrombin (FII)which are genetic risk factors for developing venous thrombosis.

The term “typing” as used herein includes any method of analysing thenucleotide sequence of the nucleic acid molecule to be analysed (i.e.the “test” or target nucleic acid). More particularly, the typing methodof the invention includes methods for detecting, identifying oranalysing genetic or sequence variation (e.g. genomic variation) in atarget nucleic acid molecule or molecules (as mentioned above, this maybe e.g. mutation, allelic variation, polymorphisms etc.). Methods of theinvention thus include methods of identifying, differentiating ordistinguishing a nucleic acid molecule or molecules. Since the typingmethod of the invention relies on detecting genetic variation in anucleic acid molecule or molecules, it may be regarded as a method ofgenotyping. It will be understood that a nucleic acid molecule mayitself be typed and also that a given variable site within a nucleicacid molecule may be typed.

“Genotyping” according to the present invention thus involvesdetermining the genotype of the target nucleic acid molecule(s). In thecontext of this specification, the “genotype” may be regarded as theparticular combination or pattern of the genetic variations which arestudied or analysed in the method of the invention, which is exhibited(or expressed) by the nucleic acid molecule(s) in question. The genotypemay thus comprise the combination (or pattern) of particular alleles(i.e. variations) which are found at the particular loci investigated.

In other words, the genotype is a combination or pattern of multiplegenetic variations (or “variable sites”) in target nucleic acid. Thegenetic variations which comprise or make up the genotype may be thoseselected for study in the method of the invention (notwithstanding thatother genetic variations may also be present in the molecule, which arenot investigated). As mentioned above, “multiple” as used herein means 2or more (or 3,4,5,6,7,8,9,10 or more), and the genetic variations (or“variable sites”) may be polymorphisms (e.g. SNPs), insertions,deletions, mutations, hypervariable regions, variable motifs, or allelicvariations, etc. According to the methods of the invention, 2 or more,preferably 3 or more, e.g. 3-7 variable sites are investigatedsimultaneously. Unless two of such variable sites are found closetogether, e.g. with 50 nucleotides, preferably within 30 nucleotides,preferably within 20 nucleotides a separate primer will be required inorder to type each variable site. So each primer will be responsible forgenerating typing information about one or more variable sites, a primerwill therefore in effect have its ‘own’ variable site(s).

Conveniently, the target nucleic acid may be DNA, although typing of RNA(e.g. mRNA) is also within the scope of the invention. If it is desiredto type a RNA sample, the method may additionally include the step ofgenerating cDNA from the RNA template, conveniently by using reversetranscriptase. Alternatively, if desired, the primer extension reactionsmay be performed directly on the RNA template.

The target nucleic acid may thus be any nucleic acid, isolated orsynthetic, in any desired or convenient form. It may thus be genomicDNA, or isolated mRNA which may be used directly for analysis by themethod of the invention, or it may be a nucleic acid product derivedtherefrom (or corresponding thereto), e.g. by synthesis, such as cDNA asmentioned above, or an amplification product (e.g. PCR amplicon), clonesor library products etc.

The nucleic acid molecule(s) may be obtained or derived from anyconvenient source, which may be any material containing nucleic acid,and all biological and clinical samples are included as possible sourcesi.e. any cell or tissue samples of an organism, or any body fluid orpreparation derived therefrom, as well as cell cultures, cellpreparations, cell lysates etc. Environmental samples e.g. soil andwater samples or food samples are also included. The samples may befreshly prepared or they may be prior-treated in any convenient way e.g.for storage.

Representative sources of nucleic acid thus include, for example, foodsand allied products, clinical and environmental samples. However, thesource will generally be a biological sample, which may contain anyviral or cellular material, including all prokaryotic or eukaryoticcells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles.Such biological material may thus comprise all types of mammalian andnon-mammalian animal cells, plant cells, algae including blue-greenalgae, fungi, bacteria, protozoa etc. Representative sources thusinclude whole blood and blood-derived products such as plasma, serum andbuffy coat, urine, faeces, cerebrospinal fluid or any other body fluids,tissues, cell cultures, cell suspensions etc.

The nucleic acid may be provided for investigation in any convenientform and conveniently will be contained in a sample, e.g. an aqueoussample (e.g. in a buffer etc.). The nucleic acid may be prepared for thetyping method, as desired, according to techniques well known in theart, e.g. isolation, purification, cloning, copying, amplification, etc.

In carrying out the method of the invention, two or more primers(“extension primers”) are provided which bind to the target nucleic acidat a predetermined site, each primer binding site being different, sothat multiple different primer extension reactions are performed. Theextension primers are designed or selected so that their extensionproducts overlap (or comprise) a site (e.g. locus or region) of sequencevariability (i.e. genetic variation) in the target nucleic acid. Inother words, the primers bind to the target nucleic at, or near to (e.g.within 1 to 40, 1 to 20, 1 to 10, or 1 to 6 bases of), a variable site.As mentioned above, such variable sites constitute the genotype of thetarget nucleic acid.

At least two extension primers are required to carry out the method,preferably at least three. However, the number of primers may be variedaccording to choice, for example, depending on the complexity of thesystem under study, and the detail of the information it is desired toobtain. Thus, for example, 3, 4, 5, or 6, or more extension primers(e.g. 3 to 15, or 3-10) may be used.

Thus, the term “variable site” refers to a site (e.g. locus or region)of a nucleic acid molecule which can differ in different genotypes. Asdefined above, the variable site may be a polymorphism or motif etc.Nucleic acid markers used for typing normally contain bothconserved/semi-conserved and variable regions. Thus, each “type” willcomprise a region of sequence variation, wherein this region (i.e. thesequence, or base identity, at that site) can be different from othertypes. In the method of the invention, at least two potential variablesites are examined, and, when one target nucleic acid molecule is typed,said nucleic acid molecule thus contains 2 or more (i.e. multiple)variable sites. Where 2 or more target nucleic acid molecules are typed,said nucleic acid molecules thus each contain 1 or more variable sites.

It will be understood by the skilled person in the art that any desiredcombination of variable sites can be analysed by the method of theinvention. The variable sites do not have to be restricted to a singlegene, coding region, non-coding region or nucleic acid molecule, but maybe found anywhere in the target genome. It will further be understoodthat the variable site can be of any length, optionally 1 to 20nucleotides, preferably 1 to 10 nucleotides in length. Typically,however, the variable site may comprise only a single or a few (e.g.1-6, e.g. 1, 2, 3, 4, 5 or 6) nucleotides at which the sequence of thetarget nucleic acid may be variable. Thus, for example, a virus such ashepatitis C virus (HCV) may contain regions which are conserved betweensub-types, but which nonetheless contain sites which may vary betweensubtypes. Such variable sites (which may typically be 1 to 3 nucleotidesin length) may thus be used to distinguish between the various subtypes.In HCV such a conserved region containing variable sites is the 5′untranslated region (5′UTR), and this may conveniently be used in agenotyping assay method of the invention, as described further inExample 1 below.

Other microorganisms will analogously have similar such regions in theirgenomes, containing variable sites, which may similarly be used in themethod of the invention. For other typing applications e.g. typing ofpolymorphisms, regions of sequence variability, analogously containingpolymorphic sites, may similarly be identified. For example, SNPs in theRenin-Angiotensinogen-Aldosterone system (RAAS) may be assessed usingprimers position in conserved regions of the genes. The primer can beposition at or near to the SNP site. FIG. 5 shows the positioning ofthree different extension primers, wherein the 3′ end of the primer is 4bases, 5 bases or 10 bases from the SNP position. The SNP is EU6 (ACET3409C).

It will be understood that in order to perform the invention the primerbinding sites should be available in all possible variants (genotypes)of the nucleic acid molecule(s) under study. Such primer binding siteswill therefore advantageously lie in regions which are common to, orsubstantially conserved between, the different variants. This mayreadily be achieved by selecting the primer binding sites to lie inconserved/semi-conserved regions as discussed above.

The primer extension reactions conveniently may be performed bysequentially adding the nucleotides to the reaction mixture (i.e. apolymerase, and primer/template mixture). Advantageously the differentnucleotides are added in known order, and preferably in a pre-determinedorder. In a convenient embodiment of the invention described in Example1 below, the 4 different nucleotides (i.e. A, G, T and C nucleotides)are added sequentially in a predetermined order of addition. It thusforms a preferred aspect of the invention that the nucleotides are addedsequentially in a predetermined order of addition. Therefore, the orderof addition can be tailored to the nucleic acid(s) to be typed and theprimers used. It will therefore be seen that the order of addition willnot necessarily be cyclical e.g. A T G C A T G C but can be e.g. C G C TA G A.

As each nucleotide is added, it may be determined whether or notnucleotide incorporation takes place.

Advantageously, as described in more detail below, it may further bedetermined the amount (i.e. how many) of each nucleotide incorporated.In this manner, the pattern of nucleotide incorporation may bedetermined. In other words, the step of determining the pattern ofnucleotide incorporation may comprise determining (or detecting) whetheror not, and which, nucleotide is incorporated. Advantageously, this stepalso includes determining the amount of each nucleotide incorporated.Such a quantitative embodiment, wherein nucleotide incorporation isdetermined quantitatively, represents a preferred aspect of theinvention.

In this manner, a “pattern” or “fingerprint” may be obtained for thetarget nucleic acid. This pattern comprises the base identity (i.e.sequence) of the particular variable sites identified for that nucleicacid molecule. In other words, the pattern corresponds to the genotypeof the target nucleic acid. The genotype may thus readily be identifiedby comparing the pattern obtained to a reference pattern (or a “standardpattern”), or a panel of reference patterns (i.e. one or more, e.g. twoor more e.g. 1 to 20, 1 to 15, 1 to 10, 1 to 6 or 1 to 3). A referencepattern may readily be obtained by determining the pattern of nucleotideincorporation using the extension primers in question on referencenucleic acid molecules of known genotype (e.g. a known microbial subtypeor a known polymorphic pattern).

Alternatively, the ‘reference pattern’ can be theoretically derived fromknowledge of the variable sites, as shown in the later Examples. It maythen not be necessary actually to compare the pattern obtained with areference pattern, the desired typing/sequence information can be readfrom the pattern obtained. Once the extension primers for each variablesite have been selected and the order of addition of nucleotidesdetermined, it is possible to determine a theoretical output from aprimer extension reaction. FIG. 6 shows the theoretical output fromsequencing two variable sites individually, and the combination ofextending both extension primers simultaneously. The theoreticalreference pattern is shown for 2 variable sites present asheterozygotes. The primers used bound 3′ to the sequences shown.

Thus, by identifying (or recognising) the pattern obtained for a targetnucleic acid molecule, the genotype of the molecule may be identified(or recognised). Conveniently, test patterns and reference patterns maybe compared using pattern recognition software.

In order to perform the invention, it may be advantageous or convenientfirst to amplify the nucleic acid molecule by any suitable amplificationmethod known in the art. The target nucleic acid would then be anamplicon. Suitable in vitro amplification techniques include any processwhich amplifies the nucleic acid present in the reaction under thedirection of appropriate primers. The amplicon method may thuspreferably be PCR, or any of the various modifications thereof e.g. theuse of nested primers, although it is not limited to this method. Thoseskilled in the art will appreciate that other amplification proceduresmay also be used, such as Self-sustained Sequence Replication (3SR),NASBA, the Q-beta replicase amplification system and Ligase chainreaction (LCR) (see for example Abramson and Myers (1993) CurrentOpinion in Biotech., 4: 41-47).

If PCR is used to amplify the nucleic acid, suitable primers, asdiscussed previously, are designed to ensure that the region of interestwithin the nucleic acid sequence (i.e. the region containing thevariable sites), is amplified. PCR can also be used for indiscriminateamplification of all DNA sequences, allowing amplification ofessentially all sequences within the sample for study (i.e. total DNA).Linker-primer PCR is particularly suitable for indiscriminateamplification, and uses double stranded oligonucleotide linkers with asuitable overhanging end, which are ligated to the ends of target DNAfragments. Amplification is then conducted using oligonucleotide primerswhich are specific for the linker sequences. Alternatively, completelyrandom oligonucleotide primers may be used in conjunction with DOP-PCR(degenerate oligonucleotide-primed) to amplify all the DNA within asample. If the variant sites to be typed by the method of the inventionare present in discrete areas of the genome, multiplex PCR can be usedto amplify nucleic acid sequences from the genome containing thevariable sites. Therefore, multiple fragments can be amplified in asingle PCR reaction.

In the method of the invention, several sequences may need to beamplified, to allow several regions (e.g. containing different variablesites) to be analysed. Therefore, several appropriate amplificationprimers may need to be synthesized to allow the selective amplificationof several sequences in the target nucleic acid. It will therefore beunderstood that a number of different nucleic acid molecules may bepresent in the reaction mixture.

One or more of the amplification primers used in the amplificationreaction, may be subsequently used as an “extension primer”, but thiswill preferably be a different primer.

It will be appreciated that the sequence and length of theoligonucleotide amplification and extension primers to be used in theamplification and extension steps, respectively, will depend on thesequence of the target nucleic acid, the desired length of amplificationor extension product, the further functions of the primer (i.e. forimmobilization) and the method used for amplification and/or extension.Appropriate primers may readily be designed applying principles andtechniques well known in the art.

Advantageously, as mentioned above, extension primers will bind near(e.g. within 1-40, 1-20, 1-10 or 1-6, preferably within 1-3 bases),substantially adjacent or exactly adjacent to the variable site of thetarget nucleic acid and will be complementary to a conserved orsemi-conserved region of the nucleic acid. In certain embodiments, asdescribed in Example 1 for instance, all primers will bind substantiallyadjacent to variable sites within the target nucleic acid (i.e. adjacentor within 3 bases of the variable site). In other embodiments, see forexample Example 3, the primers will be staggered so that one is veryclose to its variable site, another is some distance away, e.g. 4-10nucleotides distant and a third primer is 7 or more e.g. 8-16nucleotides distant from its (first) variable site. FIG. 7 depicts thisprinciple.

In order for the method of the invention to be performed, knowledge ofthe sequence of the conserved or semi-conserved region is required inorder to design an appropriate complementary extension primer. Anextension primer is provided for each of the variable regions, eachbeing specific for a site at or near to the variable site. Thespecificity is achieved by virtue of complementary base pairing. For allembodiments of the invention, primer design may be based upon principleswell known in the art. It is not necessary for the extension oramplification primer to have absolute complementarily to the bindingsite, but this is preferred to improve the specificity of binding.

The extension primer may be designed to bind to the sense or anti-sensestrand of the target nucleic acid.

In a preferred embodiment of the invention, the extension primers aredesigned to bind to the target nucleic acid near to the variable sitesin such a way that upon the addition of nucleotides in a predeterminedmanner, the typing of each variable site takes place discretely. Thusanalysis of a given variable site is not complicated by a positiveincorporation signal from other variable or conserved regions. As shownin Example 1, it is possible to interpret the test pattern and allow forsignals from nucleotide incorporation at more than the primer, butpreferably when one primer is extending over a variable site, the otherprimers will be silent. Thus, if nucleotide incorporation takes place atone variable site, there is preferably no nucleotide incorporation atthe other variable site(s). For example in the theoretical pattern shownin FIG. 6, the extension primers are positioned in such a way that, uponthe pre-determined sequential addition of nucleotides, each variablesite is typed discretely, even though primer extension-occurssimultaneously at other points—e.g. the second dispensation ofnucleotide A. In this preferred embodiment, only the variable site issequenced when an extended primer reaches its variable site; the otherprimers are not extended as the nucleotide added to the reaction mixtureis not complementary to the next base in the templates for the otherprimer-extension reactions. Thus, the primers should be designed inparallel with the order of addition of the nucleotides. Primer designsoftware can be used to determine the actual sequence of the primer oncethe 3′ end has been fixed. Preferably, Pyrosequencing Primer DesignSoftware is used.

FIG. 7 shows a simplified set of multiplex primer extension reactionswherein the extension primers are placed at differing distances from thevariant site (shown as X). This enables a pre-determined pattern ofnucleotide addition to be performed wherein the variant site issequenced in isolation. As can be envisaged, variations in thenucleotide sequence upstream of the different variable sites may meanthat the primers need not anneal in such a staggered manner but thepattern of nucleotide addition alone may be sufficient to ensure the(extended) primers approach and/or sequence their variable sites atdifferent times. Thus all primers may anneal at similar distances fromthe variable sites (or where one primer extension reaction is used totype 2 variable sites, the first variable site) e.g. substantiallyadjacent thereto, but where the nucleotides immediately upstream of andwithin the variable sites vary, so the order of nucleotide addition willcontrol the order of primer extension across the variable sites.

The “primer extension” reaction according to the invention includes allforms of template-directed polymerase-catalysed nucleic acid synthesisreactions. Conditions and reagents for primer extension reactions arewell known in the art, and any of the standard methods, reagents andenzymes etc. may be used in this step (see e.g. Sambrook et al., (eds),Molecular Cloning: a laboratory manual (1989), Cold Spring HarborLaboratory Press). Thus, the primer extension reaction at its mostbasic, is carried out in the presence of primer, deoxynucleotides(dNTPs) and a suitable polymerase enzyme e.g. T7 polymerase, Klenow orSequenase Ver 2.0 (USB USA), or indeed any suitable available polymeraseenzyme. As mentioned above, for an RNA template, reverse transcriptasemay be used. Conditions may be selected according to choice, havingregard to procedures well known in the art.

The primer is thus subjected to a primer-extension reaction in thepresence of a nucleotide, whereby the nucleotide is only incorporated ifit is complementary to the base immediately adjacent (3′) to the primerposition. The nucleotide may be any nucleotide capable of incorporationby a polymerase enzyme into a nucleic acid chain or molecule. Thus, forexample, the nucleotide may be a deoxynucleotide (dNTP, deoxynucleosidetriphosphate) or dideoxynucleotide (ddNTP, dideoxynucleosidetriphosphate). Thus, the following nucleotides may be used in theprimer-extension reaction: guanine (G), cytosine (C), thymine (T) oradenine (A) deoxy- or dideoxy-nucleotides. Therefore, the nucleotide maybe dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidinetriphosphate), dTTP (deoxythymidine triphosphate) or DATP(deoxyadenosine triphosphate). As discussed further below, suitableanalogues of dATP, and also for dCTP, dGTP and dTTP may also be used.Modified nucleotides which include an activation or detectable group,radio or fluoroscently labelled nucleotide triphosphates can also beused in the primer extension step. Dideoxynucleotides may also be usedin the primer-extension reaction. The term “dideoxynucleotide” as usedherein includes all 2′-deoxynucleotides in which the 3′ hydroxyl groupis modified or absent. Dideoxynucleotides are capable of incorporationinto the primer in the presence of the polymerase, but cannot enter intoa subsequent polymerisation reaction, and thus function as a “chainterminator”.

If the nucleotide is complementary to the target base, the primer isextended by one nucleotide, and inorganic pyrophosphate is released. Asdiscussed further below, in a preferred method, the inorganicpyrophosphate may be detected in order to detect the incorporation ofthe added nucleotide. For some variable sites, the addition of onenucleotide will be sufficient to generate typing information. However,for the majority of variable sites, data for several adjacentnucleotides will be necessary. The extended primer can serve in exactlythe same way in a repeated procedure to determine the next base in thevariable region, thus permitting the whole variable site to besequenced. Different nucleotides may be added sequentially,advantageously in known order, as discussed above, to reveal thenucleotides which are incorporated for each extension primer.Furthermore, in the case where the variable site is homopolymeric (i.e.contains 2 or more identical bases), the number of nucleotidesincorporated of the complementary base will reflect the number presentin the homopolymeric region. Accordingly, determining the number ofnucleotides incorporated for each nucleotide addition, will reveal thisinformation, and hence contribute to the pattern of nucleotideincorporation.

Hence, a primer extension protocol may involve annealing a primer asdescribed above, adding a nucleotide, performing a polymerase-catalysedprimer extension reaction, detecting the presence or absence ofincorporation of said nucleotide (and advantageously also determiningthe amount of each nucleotide incorporated) and repeating the nucleotideaddition and primer extension steps etc. one or more times. As discussedabove, single (i.e. individual) nucleotides may be added successively tothe same primer-template mixture, or to separate aliquots ofprimer-template mixture, etc. according to choice.

In order to permit the repeated or successive (iterative) addition ofnucleotides in a primer-extension procedure, the previously-addednucleotide must be removed. This may be achieved by washing, or moreconveniently, by using a nucleotide-degrading enzyme, for example asdescribed in detail in WO98/28440.

Accordingly, in a principal embodiment of the present invention, anucleotide degrading enzyme is used to degrade any unincorporated orexcess nucleotide. Thus, if a nucleotide is added which is notincorporated (because it is not complementary to the target base), orany added nucleotide remains after an incorporation event (i.e. excessnucleotides) then such unincorporated nucleotides may readily be removedby using a nucleotide-degrading enzyme. This is described in detail inWO98/28440.

The term “nucleotide degrading enzyme” as used herein includes anyenzyme capable of specifically or non-specifically degradingnucleotides, including at least nucleoside triphosphates (NTPs), butoptionally also di- and mono-phosphates, and any mixture or combinationof such enzymes, provided that a nucleoside triphosphatase or otherNTP-degrading activity is present. Where a chain terminating nucleotideis used (e.g. a dideoxy nucleotide is used), the nucleotide degradingenzyme should also degrade such a nucleotide. Althoughnucleotide-degrading enzymes having a phosphatase activity mayconveniently be used according to the invention, any enzyme having anynucleotide or nucleoside degrading activity may be used, e.g. enzymeswhich cleave nucleotides at positions other than at the phosphate group,for example at the base or sugar residues. Thus, a nucleosidetriphosphate degrading enzyme is essential for the invention. Nucleosidedi- and/or mono-phosphate degrading enzymes are optional and may be usedin combination with a nucleoside tri-phosphate degrading enzyme.

The preferred nucleotide degrading enzyme is apyrase, which is both anucleoside diphosphatase and triphosphatase, catalysing the reactionsNTP→NDP+Pi and NDP→NMP+Pi (where NTP is a nucleoside triphosphate, NDPis a nucleoside diphosphate, NMP is a nucleotide monophosphate and Pi isinorganic phosphate). Apyrase may be obtained from the Sigma ChemicalCompany. Other possible nucleotide degrading enzymes include PigPancreas nucleoside triphosphate diphosphorydrolase (Le Bel et al.,1980, J. Biol. Chem.,255, 1227-1233). Further enzymes are described inthe literature.

The nucleotide-degrading enzyme may conveniently be included during thepolymerase (i.e. primer extension) reaction step. Thus, for example thepolymerase reaction may conveniently be performed in the presence of anucleotide-degrading enzyme. Although less preferred, such an enzyme mayalso be added after nucleotide incorporation (or non-incorporation) hastaken place, i.e. after the polymerase reaction step.

Thus, the nucleotide-degrading enzyme (e.g. apyrase) may be added to thepolymerase reaction mixture (i.e. target nucleic acid, primer andpolymerase) in any convenient way, for example prior to orsimultaneously with initiation of the reaction, or after the polymerasereaction has taken place, e.g. prior to adding nucleotides to thesample/primer/polymerase to initiate the reaction, or after thepolymerase and nucleotide are added to the sample/primer mixture.

Conveniently, the nucleotide-degrading enzyme may simply be included inthe reaction mixture for the polymerase reaction, which may be initiatedby the addition of the nucleotide.

According to the present invention, detection of nucleotideincorporation can be performed in a number of ways, such as byincorporation of labelled nucleotides which may subsequently bedetected, or by using labelled probes which are able to bind to theextended sequence.

The method may be performed using a sanger sequencing method combinedwith a standard detection strategy, e.g. electrophoresis or massspectometry to analyse, or determine, nucleotide incorporation. However,it is preferred to use a sequencing-by-synthesis method, due to the factthat the extension reactions are quantitative, i.e. that the nucleotideincorporation may be determined quantitatively. As mentioned above,sequencing-by-synthesis methods are disclosed extensively in U.S. Pat.No. 4,863,849, which discloses a number of ways in which activatednucleotide incorporation may be determined or detected, e.g.spectrophotometrically or by fluorescent detection techniques, forexample by determining the amount of nucleotide remaining in the addednucleotide feedstock, following the nucleotide incorporation step. In asequencing-by-synthesis reaction, determination of the pattern ofnucleotide incorporation occurs simultaneously with primer extension.One working definition of Sequencing by synthesis is a method in which asingle activated (i.e. labelled)nucleotide is or is not incorporatedinto a primed template, incorporation being detected by any suitablemeans. This step is repeated by addition of a different activatednucleotide and incorporation is again detected. These steps are repeatedand from the sum of incorporated nucleic acids the sequence can bededuced. The preferred method of sequencing-by-synthesis is however apyrophosphate detection-based method.

Preferably, therefore, nucleotide incorporation is detected by detectingPPi release, preferably by luminometric detection, and especially bybioluminometric detection.

PPi can be determined by many different methods and a number ofenzymatic methods have been described in the literature (Reeves et al.,(1969), Anal. Biochem., 28, 282-287; Guillory et al., (1971), Anal.Biochem., 39, 170-180; Johnson et al., (1968), Anal. Biochem., 15, 273;Cook et al., (1978), Anal. Biochem. 91, 557-565; and Drake et al.,(1979), Anal. Biochem. 94, 117-120).

It is preferred to use luciferase and luciferin in combination toidentify the release of pyrophosphate since the amount of lightgenerated is substantially proportional to the amount of pyrophosphatereleased which, in turn, is directly proportional to the amount ofnucleotide incorporated. The amount of light can readily be estimated bya suitable light sensitive device such as a luminometer. Thus,luminometric methods offer the advantage of being able to bequantitative.

Luciferin-luciferase reactions to detect the release of PPi are wellknown in the art. In particular, a method for continuous monitoring ofPPi release based on the enzymes ATP sulphurylase and luciferase hasbeen developed (Nyrén and Lundin, Anal. Biochem., 151, 504-509, 1985;Nyrén P., Enzymatic method for continuous monitoring of DNA polymeraseactivity (1987) Anal. Biochem Vol 167 (235-238)) and termed ELIDA(Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay). Theuse of the ELIDA method to detect PPi is preferred according to thepresent invention. The method may however be modified, for example bythe use of a more thermostable luciferase (Kaliyama et al., 1994,Biosci. Biotech. Biochem., 58, 1170-1171) and/or ATP sulfurylase (Ondaet al., 1996, Bioscience, Biotechnology and Biochemistry, 60:10,1740-42). This method is based on the following reactions:

Reference may also be made to WO 98/13523 and WO 98/28448, which aredirected to pyrophosphate detection-based sequencing procedures, anddisclose PPi detection methods which may be of use in the presentinvention.

In a PPi detection reaction based on the enzymes ATP sulphurylase andluciferase, the signal (corresponding to PPi released) is seen as light.The generation of the light can be observed as a curve known as aPyrogram™. Light is generated by luciferase action on the product, ATP(produced by a reaction between PPi and APS (see below) mediated by ATPsulphurylase) and, where a nucleotide-degrading enzyme such as apyraseis used, this light generation is then “turned off” by the action of thenucleotide-degrading enzyme, degrading the ATP which is the substratefor luciferase. The slope of the ascending curve may be seen asindicative of the activities of DNA polymerase (PPi release) and ATPsulphurylase (generating ATP from the PPi, thereby providing a substratefor luciferase). The height of the signal is dependent on the activityof luciferase, and the slope of the descending curve is, as explainedabove, indicative of the activity of the nucleotide-degrading enzyme. Asexplained below, Pyrogram™ in the context of a homopolymeric region,peak height is also indicative of the number of nucleotides incorporatedfor a given nucleotide addition step. Then, when a nucleotide is added,the amount of PPi released will depend upon how many nucleotides (i.e.the amount) are incorporated, and this will be reflected in the slopeheight.

Advantageously, by including the PPi detection enzyme(s) (i.e. theenzyme or enzymes necessary to achieve PPi detection according to theenzymatic detection system selected, which in the case of ELIDA, will beATP sulphurylase and luciferase) in the polymerase reaction step, themethod of the invention may readily be adapted to permit extensionreactions to be continuously monitored in real-time, with a signal beinggenerated and detected, as each nucleotide is incorporated.

Thus, the PPi detection enzymes (along with any enzyme substrates orother reagents necessary for the PPi detection reaction) may simply beincluded in the polymerase reaction mixture.

A potential problem which has previously been observed with PPi-basedsequencing methods is that DATP, used in the chain extension reaction,interferes in the subsequent luciferase-based detection reaction byacting as a substrate for the luciferase enzyme. This may be reduced oravoided by using, in place of deoxyadenosine triphosphate (ATP), a DATPanalogue which is capable of acting as a substrate for a polymerase butincapable of acting as a substrate for a PPi-detection enzyme. Such amodification is described in detail in WO98/13523.

The term “incapable of acting” includes also analogues which are poorsubstrates for the detection enzymes, or which are substantiallyincapable of acting as substrates, such that there is substantially no,negligible, or no significant interference in the PPi detectionreaction.

Thus, a further preferred feature of the invention is the use of a DATPanalogue which does not interfere in the enzymatic PPi detectionreaction but which nonetheless may be normally incorporated into agrowing DNA chain by a polymerase. By “normally incorporated” is meantthat the nucleotide is incorporated with normal, proper base pairing. Inthe preferred embodiment of the invention where luciferase is a PPidetection enzyme, the preferred analogue for use according to theinvention is the [1-thioltriphosphate (or α-thiotriphosphate) analogueof deoxy ATP, preferably deoxyadenosine [1-thio]triphospate, ordeoxyadenosine α-thiotriphosphate (dATPαS) as it is also known. dATPαS,along with the α-thio analogues of dCTP, dGTP and dTTP, may be purchasedfrom Amersham Pharmacia. Experiments have shown that substituting dATPwith dATPαS allows efficient incorporation by the polymerase with a lowbackground signal due to the absence of an interaction between dATPαSand luciferase. False signals are decreased by using a nucleotideanalogue in place of dATP, because the background caused by the abilityof dATP to function as a substrate for luciferase is eliminated. Inparticular, an efficient incorporation with the polymerase may beachieved while the background signal due to the generation of light bythe luciferin-luciferase system resulting from DATP interference issubstantially decreased. The dNTPαS analogues of the other nucleotidesmay also be used in place of the other dNTPs.

Another potential problem which has previously been observed withsequencing-by-synthesis methods is that false signals may be generatedand homopolymeric stretches (i.e. CCC) are difficult to sequence withaccuracy. This may be overcome by the addition of a single-strandednucleic acid binding protein (SSB) once the extension primers have beenannealed to the template nucleic acid. The use of SSB insequencing-by-synthesis is discussed in WO 00/43540 of PyrosequencingAB.

It will be understood that in the method of the invention, differingamounts of nucleic acid template may be present when multiple nucleicacid molecules are to be typed. In order to be able to quantify thenumber of nucleotides incorporated upon addition in certain embodiments,it is preferred to design the primers and nucleotide dispensation insuch a way that a reference signal is generated for each primer whichcorresponds to a single nucleotide incorporation event. The referencesignal is generated in the absence of nucleotide incorporation in theother primer-extension reactions. The reference signal allows forcalibration of the signals relating to the same template. The referencepeaks are clearly shown on FIG. 9, and the height of the variant sitesignal can be correlated to the reference signal to increase accuracy.

The step of detecting nucleotide incorporation by detecting PPi releaseresults in a signal indicative of the amount of pyrophosphate released,and hence the amount of nucleotide incorporated. In the method of theinvention, 2 or more distinct primers are used sequentially orsimultaneously in a primer-extension reaction. Thus, in the case of thesimultaneously added primers, for every nucleotide addition, 0, 1 ormore nucleotides may be incorporated into the growing DNA chains. Thesignal generated in the pyrophosphate detection step will therefore beindicative of the number of nucleotides incorporated in theprimer-extension step for the combination of all primers bound to thetemplate DNA. The size of the signal (i.e. the height of each peak) cantherefore be correlated directly to the number of incorporatednucleotides. In certain embodiments, the primer needs only to besubjected to 1 to 20, preferably 1 to 10, e.g. 1 to 5 and mostpreferably 1 to 4 cycles of nucleotide addition.

In one embodiment of the invention, 2 or more primers are hybridized(simultaneously) at, adjacent or near to variable sites in the targetnucleic acid. Each primer being responsible for the-typing of one orpossibly more variable sites. Primer extension is then performed asdescribed above, and primer extension occurs for each primer only if thenucleotide added is complementary to the target base. Thus, when 2primers are used simultaneously, none, 1, 2 or more (for homopolymericregions) nucleotide incorporation events may occur upon the addition ofany given nucleotide. The primer extension reaction is carried outsimultaneously for all hybridized primers in the reaction mixture. Thus,the detected nucleotide incorporation gives a cumulative picture for allhybridized primers. In this manner, the pattern of nucleotideincorporation may be directly determined. Preferably, when an extensionreaction extends across a variable site, nucleotide incorporation occursonly at that site.

In a further embodiment of the invention, the primers may be addedsequentially to the primer extension reaction.

In this case, the pattern of nucleotide incorporation may be determinedfor each primer separately, and then “added together” to obtain acumulative picture/pattern. In a modified version of this embodiment ofthe invention, the first primer is hybridized to the target nucleicacid, undergoes a primer extension reaction, which is terminated afterthe variable site has been sequenced, by the addition of a chainterminator. Chain terminators are well known in the art, and includedideoxynucleotides. A second primer is then added to sequence a secondvariable site, and the sequencing is again terminated by the addition ofa chain terminator. This method may be repeated until all variableregions of interest have been sequenced.

In a further particularly preferred embodiment which is also discussedabove and in the Examples, the extension primers are hybridized to thetemplate, and the primers are extended simultaneously. The primers aredesigned to enable primer extension to occur over the variable sitessequentially—i.e. primer extension occurs for each primersimultaneously, but primer extension over a variable site occurs inturn, whilst the other primers are extended over aconserved/semiconserved region or more preferably are not extended atall due to the addition of non-complementary nucleotides. The pattern ofnucleotide addition is preferably pre-determined to allow extension ofthe primers to occur sequentially over the variable sites. The primersmay bind 1 to 40, 1 to 20, 1 to 10, 1 to 5 nucleotides from or adjacentto the variable site.

Optionally once a primer has been extended over a variable site, a chainterminator, such as a dideoxynucleotide, may be added to specificallyterminate the chain extension reaction of that primer. It will beunderstood that nucleotide incorporation signals will be generated forall primers during the primer extension reaction, and will contribute tothe pattern obtained. Nevertheless, different regions of the patternwill preferably relate to just one of the variable sites.

In a still further modified embodiment of the invention, chainterminators may be employed in place of dNTPs or in combination withdNTPs, using simultaneously hybridised primers. In this case, theprimers are selected or designed to ensure that primer extension fromeach primer takes place sequentially, i.e. that nucleotides are firstincorporated from the first primers, the first extension reaction iscomplete, before nucleotide incorporation from the next primer takesplace. This embodiment also requires that the nucleotides are added inpredetermined order.

Indeed, so-called “intelligent” primer design may be used to carry outthe method of the invention in a desired or pre-selected (i.e.predetermined) manner. This may be applied both to the number ofextension primers employed, and to the design of the sequence thereof.“Intelligent” primer design is optimally performed with an “intelligent”order of addition of nucleotides to enable the sequencing of theindividual variable sites to be performed in isolation. Such‘intelligent’ design of primers and the order of nucleotide addition isdescribed in more detail in the Examples.

The method of the invention may conveniently be performed in a singlereaction vessel, whether a “simultaneous” or “sequential” primerextension embodiment is used. Thus, for example, all extension primersmay be added together, or sequentially into a single reaction vessel.

In order for the primer-extension reaction to be performed, the nucleicacid molecule, regardless of whether or not it has been amplified, isconveniently provided in a single-stranded format. The nucleic acid maybe subjected to strand separation by any suitable technique known in theart (e.g. Sambrook et al., supra), for example by heating the nucleicacid, or by heating in the presence of a chemical denaturant such asformamide, urea or formaldehyde, or by use of alkali.

However, this is not absolutely necessary and a double-stranded nucleicacid molecule may be used as template, e.g. with a suitable polymerasehaving strand displacement activity.

Where a preliminary amplification step is used, regardless of how thenucleic acid has been amplified, all components of the amplificationreaction need to be removed, to obtain pure nucleic acid, prior tocarrying out the typing assay of the invention. For example,unincorporated nucleotides, PCR primers, and salt from a PCR reactionneed to be removed. Methods for purifying nucleic aids are well known inthe art (Sambrook et al., supra), however a preferred method is toimmobilize the nucleic acid molecule, removing the impurities viawashing and/or sedimentation techniques.

Optionally, therefore, the target nucleic acid may be provided with ameans for immobilization, which may be introduced during amplification,either through the nucleotide bases or the primer/s used to produce theamplified nucleic acid.

To facilitate immobilization, the amplification primers used accordingto the invention may carry a means for immobilization either directly orindirectly. Thus, for example the primers may carry sequences which arecomplementary to sequences which can be attached directly or indirectlyto an immobilizing support or may carry a moiety suitable for direct orindirect attachment to an immobilizing support through a bindingpartner.

Numerous suitable supports for immobilization of DNA and methods ofattaching nucleotides to them, are well known in the art and widelydescribed in the literature. Thus for example, supports in the form ofmicrotitre wells, tubes, dipsticks, particles, fibres or capillaries maybe used, made for example of agarose, cellulose, alginate, teflon, latexor polystyrene. Advantageously, the support may comprise magneticparticles e.g. the superparamagnetic beads produced by Dynal AS (Oslo,Norway) and sold under the trademark DYNABEADS. Chips may be used assolid supports to provide miniature experimental systems as describedfor example in Nilsson et al. (Anal. Biochem. (1995), 224:400-408).

The solid support may carry functional groups such as hydroxyl,carboxyl, aldehyde or amino groups for the attachment of the primer orcapture oligonucleotide. These may in general be provided by treatingthe support to provide a surface coating of a polymer carrying one ofsuch functional groups, e.g. polyurethane together with a polyglycol toprovide hydroxyl groups, or a cellulose derivative to provide hydroxylgroups, a polymer or copolymer of acrylic acid or methacrylic acid toprovide carboxyl groups or an amino alkylated polymer to provide aminogroups. U.S. Pat. No. 4,654,267 describes the introduction of many suchsurface coatings.

Alternatively, the support may carry other moieties for attachment, suchas avidin or streptavidin (binding to biotin on the nucleotidesequence), DNA binding proteins (e.g. the lac I repressor proteinbinding to a lac operator sequence which may be present in the primer oroligonucleotide), or antibodies or antibody fragments (binding tohaptens e.g. digoxigenin on the nucleotide sequence). Thestreptavidin/biotin binding system is very commonly used in molecularbiology, due to the relative ease with which biotin can be incorporatedwithin nucleotide sequences, and indeed the commercial availability ofbiotin-labelled nucleotides. This represents one preferred method forimmobilisation of target nucleic acid molecules according to the presentinvention. Streptavidin-coated DYNABEADS are commercially available fromDynal AS.

As mentioned above, immobilization may conveniently take place afteramplification. To facilitate post amplification immobilisation, one orboth of the amplification primers are provided with means forimmobilization. Such means may comprise as discussed above, one of apair of binding partners, which binds to the corresponding bindingpartner carried on the support. Suitable means for immobilization thusinclude biotin, haptens, or DNA sequences (such as the lac operator)binding to DNA binding proteins.

When immobilization of the amplification products is not performed, theproducts of the amplification reaction may simply be separated by forexample, taking them up in a formamide solution (denaturing solution)and separating the products, for example by electrophoresis or byanalysis using chip technology. Immobilization provides a ready andsimple way to generate a single-stranded template for the extensionreaction. As an alternative to immobilization, other methods may beused, for example asymmetric PCR, exonuclease protocols or quickdenaturation/annealing protocols on double stranded templates may beused to generate single stranded DNA. Such techniques are well known inthe art.

The method of the invention allows the typing (e.g. genotyping) of oneor more nucleic acid molecule derived from an individual (e.g. a patientunder clinical test, a tissue sample for typing, or a microorganism foridentification). Thus, the method of the invention is capable ofdistinguishing between different genotypes within a species. This isparticularly useful in the field of identification of microbial species,where many genotypes of one microbe may exist, for example, there arecurrently seven known genotypes of the Hepatitis C Virus.

The method of the present invention is particularly advantageous in thediagnosis of pathological conditions characterised by the presence ofspecific DNA, particularly latent infectious diseases such as viralinfection e.g. by herpes, hepatitis or HIV. Also, the method can be usedto characterise or type and quantify bacterial, protozoal and fungalinfections where samples of an injecting organism may be difficult toobtain or where an isolated organism is difficult to grow in vitro forsubsequent characterisation as in the case of P. falciparum or Chlamydiaspecies. Due to the simplicity and speed of the method it may also beused to detect other pathological agents which cause diseases such assyphilis and meningitis. Even in cases where samples of the injectingorganism may be easily obtained, the speed of this method compared withovernight incubation of a culture may make the method according to theinvention preferable over conventional techniques.

The method of the present invention may be used to analyse two or moresingle nucleotide polymorphisms (SNPs) within one or more genes, or twoor more genes, in an individual. Many diseases and conditions may beassociated with (or linked to) combinatorial polymorphisms within thesame gene, or within distinct genes. For example, in WO 00/22166, it hasbeen suggested that a combination of SNPs within several genes gives apolymorphic pattern which may be used to predict the likelihood ofcardiovascular disease, allowing detailed prognosis for an individual,and predicting whether a particular therapeutic regime would beeffective in improving a cardiovascular condition. Thus, the method ofthe invention can be used to give a quick prognosis on the particulargenotype of an individual, allowing tailored therapy to be administered.Example 2 shows that multiplex genotyping can be performed for SNPs inthe RAAS system. In this example, one nucleic acid contains 2 SNPs (EU7)and two additional nucleic acids contain 1 SNP each (EU8 and EU11).

The method of the invention is advantageous in that it determines theexact sequence of the variable sites (i.e. is based on a sequencingprocedure, it avoids costly and cumbersome procedures, such aselectrophoresis, and advantageously labelled nucleotides and/or primers,and large numbers of samples can be analysed in a short time.

The primer extension reaction generates a “pattern” or “fingerprint”indicative of nucleotide incorporation, correlated to the nucleotideadded to the reaction mixture. The pattern is a cumulative picture ofnucleotide incorporation for the primers designed to detect nucleotideincorporation at 2 or more variable sites within the target nucleic acidmolecule(s). To enable the target nucleic acid molecule(s) to be typed,reference patterns are used, using the same variable sites and extensionprimers. Each genotype should produce a different pattern, facilitatingidentification by comparison to the reference pattern which can bedetermined theoretically.

The method of the invention relies upon the knowledge of the locationand nature of the variable sites, together with further known sequenceinformation (e.g. with known sequences of conserved/semi-conservedregions) from which to determine an appropriate primer binding site anddesign a complementary extension primer. Using the method of theinvention, any combination of variable sites may be used in the typingmethod. It will be understood by those skilled in the art that themethod of the invention is not limited to multiple variable sites withingenes, but the method is also applicable to non-coding regions. Thepattern may be obtained for variable sites which are in one or more ofthe same gene, in related genes, in disparate genes, or in non-codingregions.

The invention also comprises kits for carrying out the method of theinvention. These will normally include one or more of the followingcomponents:

-   -   optionally primer(s) for in vitro amplification; two or more        primers for the primer extension reaction; nucleotides for        amplification and/or for the primer extension reaction (as        described above); a polymerase enzyme for the amplification        and/or primer extension reaction; and means for detecting primer        extension (e.g. means of detecting the release of pyrophosphate        as outlined and defined above).

In certain embodiments, the kit will also include instructions for theorder of addition of the nucleotides.

The invention will now be described by way of non-limiting examples withreference to the drawings in which:

FIG. 1 shows schematically one method for the typing of nucleic acidusing multiple primers (multiplexing) simultaneously in a primerextension reaction;

FIG. 2 shows the sequence of the 5′ untranslated region (5′-UTR) ofseven Hepatitis C virus (HCV) genotypes, wherein the arrows indicate thepositions of the amplification and extension primers, and thenucleotides highlighted in bold type illustrate the variable region tobe sequenced by the primer extension reaction;

FIG. 3 shows theoretical traces which would be obtained (light generated(indicating nucleotide incorporation) versus time (and nucleotideaddition)), for the seven genotypes of HCV studied. The experimentalconditions and extension primers theoretically used are described inExample 1. Three distinct extension primers were theoretically usedsimultaneously in a primer-extension reaction mixture. Inorganicpyrophosphate PPi is released in a DNA-polymerase catalyzed reaction ifa nucleotide is incorporated. The PPi is monitored by coupled enzymaticreactions using ATP sulphurylase and luciferase. Light generated as aresult is measured by a CCD detector or luminometer;

FIG. 4 shows traces (light generated (indicating nucleotideincorporation) versus time (and nucleotide addition)), obtained for sixsamples containing different HCV genotypes. The experimental conditionsand primers used are described in Example 1. The incorporation of anucleotide into the extending primer results in the release of PPi,which is detected using a coupled enzymatic reactions using ATPsulphurylase and luciferase. Light generated as a result of successfulextension is measured by a CCD camera or luminometer;

FIG. 4 a shows the trace obtained for HCV genotype 1 a;

FIG. 4 b shows the trace obtained for HCV genotype 1 b;

FIG. 4 c shows the trace obtained for HCV genotype 2 a;

FIG. 4 d shows the trace obtained for HCV genotype 2 b;

FIG. 4 e shows the trace obtained for HCV genotype 3 a;

FIG. 4 f shows the trace obtained for HCV genotype 3 b;

FIG. 5 shows three potential primer binding positions for the SNP Eu6from the ACE gene (Angiotensin Converting Enzyme). FIG. 5 a shows aprimer (boxed nucleotides) bound to the template with its 3′ end 4nucleotides from the SNP position, FIG. 5 b shows a primer bound to thetemplate with the. 3′ and 5 nucleotides from the SNP position and inFIG. 5 c the primer is bound 10 nucleotides from the SNP position. Inall figures, the 2 potential variants at the SNP site are shown (G/A intemplate strand);

FIG. 6 shows the theoretical output from Pyrosequencing™ reactions fortwo SNP positions. The theoretical output is plotted as nucleotidedispensed into the reaction versus peak height (correlated to lightemitted from the Pyrosequencing™ reaction). FIG. 6 a shows thetheoretical output for sequencing G/ACAG, in this case, the primer wouldbe adjacent to the polymorphic position. The theoretical output shown isfor the heterozygote (i.e. the individual has one copy of the SNP A andone copy of the SNP G). FIG. 6 b shows the theoretical output forTGAAC/TA. The primer is thus bound 4 nucleotides away from the SNP.Again, the pattern shown would result from a heterozygous individual(C/T). FIG. 6 c shows a cumulation of the two individual sequencingreactions in one primer extension reaction mixture;

FIG. 7 shows a simplified multiplexing analysis wherein the extensionprimers are designed in such a way that their 3′ ends are positioned atdifferent distances from the polymorphic position. This enables thedesign of an “intelligent” order of addition of nucleotides to bedetermined to enable the SNP (marked X) to be sequenced in isolation.Thus, the extension primers should be designed in parallel with thedispensation order;

FIG. 8 shows the theoretical output for five SNPs present in the RAASsystem—Eu4 (ACE G2215A), Eu8 (ATG C521T), Eu10 (ATP T573C), Eu6 (ACET3409C) and Eu3 (ACE T1237C). The theoretical outputs are plotted as inFIG. 7. The extension primers are positioned such that the sequence thatis analysed for Eu4 is G/A CTGCCTG, Eu8 is CACCA/GTGG, Eu10 isC/TCCGATAGGGC, Eu6 is ACTTC/TG and Eu3 is AGACA/GGGC;

FIGS. 8 a and 8 b show the theoretical output expected to be obtainedwhen the SNPs Eu4 and Eu8 are typed in a standard one-primer onlyreaction. The SNP nucleotide incorporation position are framed;

FIG. 8 a shows the theoretical output when the individual isheterozygous (A/G) and FIG. 8 b shows the output expected when theindividual is homozygous (G/G). FIG. 8 c shows the output expected whenthe two SNPs are sequenced simultaneously in the same reaction(multiplexed). The polymorphic positions are framed;

FIGS. 8 d, 8 e and 8 f are the theoretical results obtained fromsequencing Eu10, Eu6 and Eu3 alone, respectively. SNP Eu10 and Eu6 areshown as heterozygotes (C/T and C/T, respectively) and Eu3 as ahomozygote (A/A). The theoretical patterns for the 3 SNPs are combinedin FIG. 8 g, and the SNP positions are framed;

FIG. 9 shows the results obtained as traces (light generated (indicatingnucleotide incorporation) versus time and nucleotide incorporation forseven reactions containing differing templates and primer combinations.The experimental conditions and primers used are described in Example 2.The incorporation of a nucleotide into the extending primer results inthe release of PPi, which is detected using a coupled enzymaticreactions using ATP sulphurylase and luciferase. Light generated as aresult of successful extension is measured by a CCD camera orluminometer;

FIG. 9 a shows the trace obtained for Eu4;

FIG. 9 b shows the trace obtained for Eu8;

FIG. 9 c shows the trace obtained for Eu4 and Eu8 simultaneouslysequenced;

FIG. 9 d shows the trace obtained for Eu10;

FIG. 9 e shows the trace obtained for Eu6;

FIG. 9 f shows the trace obtained for Eu3;

FIG. 9 f shows the trace obtained for Eu10, Eu6 and Eu3, simultaneouslysequenced;

FIG. 10 shows the theoretical output for SNPs Eu8, Eu7 and Eu11 presentin the RAAS system. The theoretical outputs are plotted as in FIG. 7.The sequences analyzed are Eu8 CACCA/GTGGACAG, Eu7 T/CGGCCGGGTCACGAG/TGand Eu11 GAGCA/GTTAG. Therefore, the fragment Eu7 contains twopolymorphic sites;

FIG. 10 a shows the theoretical trace for Eu8 (G/G);

FIG. 10 b shows the theoretical trace for Eu7 (C/C and T/T);

FIG. 10 c shows the theoretical trace for Eu11 (A/G); and

FIG. 10 d shows the multiplex theoretical trace for Eu7, Eu8 and Eu11;

FIG. 11 shows the result obtained as a trace (light generated versusnucleotide addition) for the multiplex reaction as defined in Example 3.The polymorphic positions are framed and the reference peaks shown(arrows). The genotype for the individual typed is Eu8 G/G, Eu7 C/C andT/T Eu11 A/G;

FIG. 12 is a scheme showing primer design for 3 separate nucleic acidfragments, for the Plasminogen Activator Inhibitor I gene, theProthrombin gene and the Factor V gene. The arrows marked with a *correspond to the sequencing primers and the outer arrows correspond tothe PCR primers. The biotinylated primer is indicated with a B. Thepolymorphic position in the gene of interest is marked by an X;

FIG. 13 shows the expected output from Pyrosequencing™ reactions forSNPs in Plasminogen-activator inhibitor 1 (4G/5G deletion), Prothrombin(G20210A) and Factor V (G1691A). The theoretical output is shown as forFIG. 7. The sequence analysed for PAI1 is (C)ACGTG, Prothrombin isGCTC/TGCTGA and Factor V AGGCA/GAGGAA;

FIG. 13 a shows the theoretical trace for PAI1 (C/C—no deletion);

FIG. 13 b shows the theoretical trace for Prothrombin (C/T);

FIG. 13 c shows the theoretical trace for Factor V (A/G);

FIG. 13 d shows the theoretical trace for a combination of the threeSNPs in one multiplex reaction;

FIG. 13 e shows the theoretical trace for the genotype PAI1 C/C (nodeletion), Prothrombin C/C and Factor V GIG;

FIG. 13 f shows the theoretical trace for the genotype PAI1 doubledeletion, Prothrombin C/C and Factor V A/A;

FIG. 13 g shows the theoretical trace for the genotype PAI1 del/C,Prothrombin T/T and Factor V G/G;

FIG. 13 h shows the theoretical trace for the genotype PAI1 C/C (nodeletion), Prothrombin T/C and Factor V G/G;

FIG. 13 i shows the theoretical trace for the genotype PAI1 C/del,Prothrombin C/C and Factor V GIG; and

FIG. 13 j shows the theoretical trace for the genotype PAI1 C/C (nodeletion), Prothrombin C/C and Factor V A/G;

FIG. 14 shows the results obtained as traces (light geneated versusnucleotide addition) for six reactions. The materials and methods aredescribed in Example 4;

FIG. 14 a shows the trace obtained for the genotype PAI1 C/C (nodeletion), Prothrombin C/C and Factor V G/G and corresponds to thetheoretical pattern shown on FIG. 13 e;

FIG. 14 b shows the trace obtained for the genotype PAI1 doubledeletion, Prothrombin C/C and Factor V A/A and corresponds to thetheoretical pattern shown on FIG. 13 f;

FIG. 14 c shows the trace-obtained for the genotype PAI1 del/C,Prothrombin T/T and Factor V G/G and corresponds to the theoreticalpattern shown on FIG. 13 g;

FIG. 14 d shows the trace obtained for the genotype PAI1 C/C (nodeletion), Prothrombin T/C and Factor V G/G and corresponds to thetheoretical pattern shown on FIG. 13 h;

FIG. 14 e shows the trace obtained for the genotype PAI1 C/del,Prothrombin C/C and Factor V G/G and corresponds to the theoreticalpattern shown on FIG. 13 i; and

FIG. 14 f shows the trace obtained for the genotype PAI1 C/C (nodeletion), Prothrombin C/C and Factor V A/G and corresponds to thetheoretical pattern shown on FIG. 13 j;

FIG. 15 depicts the localisation of the primers with regard to theCYP2D6 gene. A segment of the gene with particular highlightedpolymorphisms can be seen at the top of this figure. The 61118 and 2162fragments, as amplified by nested PCR primers are at the bottom of thefigure. The extension primers used for the multiplexing reactions areshown above the 61118 and 2162 fragments;

FIG. 16 represents the theoretical output obtained for two genotypes ofthe CYP2D6 gene. The traces were calculated as described previously;

FIG. 16 a shows the theoretical output for G1934 A (A/G), G 1749 C(C/G), T1795 del (no deletion) and G 1846 T (T/g);

FIG. 16 b shows the theoretical output for G1934 A (A/G), G 1749 C(C/G), T1795 del T/deletion and G1846T (T/G); and

FIG. 17 shows the result obtained from Example 5 as a trace (lightgenerated versus nucleotide added). The experimental conditions aredescribed in Example 5. The genotype of the individual typed is G1934AG/G, G1749C G/G, T1795 del T/T (no deletion) G1846T G/G. Also shown isthe theoretical output plot for this genotype.

EXAMPLE 1

Serum Samples

72 sera from HCV-positive Veterans were obtained from Stanford Veteranhospital. 10 HCV-positive sera were obtained from Iran.

Synthesis and Purification of Oligonucleotides

The oligonucleotides HCV-PCR-OUTF (5′-CCCTGTGAGGAACTWCTGTCTTCACGC),HCV-PCR-OUTR (5′-GCTCATGRTGCACGGTCTACGAGACCT), HCV-PCR-INF(5′-TCTAGCCATGGCGTTAGTAYGAGTGT), BHCV-PCR-INR(5′-Biotin-CACTCGCAAGCACCCTATCAGGCAGT), HCV-SEQF1(5′-GGAACCGGTGAGTACACCGGAAT), HCV-SEQF2 (5′-GACYGGGTCCTTTCTTGGA),HCV-SEQF3 (5′-ATTTGGGCGTGCCCCCGC), were all synthesized and HPLCpurified by MWG Biotech (High points, N.C., USA).

RNA Extraction, cDNA Synthesis and Amplification

RNA was extracted from 100 μl of patient sera using Ambion's Totally RNAisolation kit (www.ambion.com, Ambion (Europe) Ltd., Cambridge, UK).cDNA was synthesized using the kit Superscipt™ Preamplification systemfrom Invitrogen (www.invitrogen.com, Invitrogen Ltd., Paisley, UK).First strand cDNA synthesis employed an RNA/primer mixture containing, 5μl RNA and 1 μl 0.5 μg/μl Oligo (dT) random primer which was incubatedat 70° C. for 10 min and then placed on ice for at least 1 min. Areaction mixture contating 2 μl 10× PCR buffer (200 mM Tris-HCl (pH8.4), 500 mM KCl), 2 μl 25 mM MgCl₂ 10 mM DNTP mix and 0.1 M DTT, wasadded to each RNA/primer mixture, mixed gently collected by briefcentrifugation and then incubated at 42° C. for 5 min. Two hundred unitsof Superscript II Reverse Transcriptase was added to each tube, andincubated at 40° C. for 50 min. The reaction was terminated byincubating at 70° C. for 15 minutes and then chilled on ice. The nucleicacid was collected by brief centrifugation. 1 μl of RNase H was added toeach tube and incubated for 20 mm at 37° C. Outer PCR was performed on 1μl of cDNA using HCV-PCR-OUTF and HCV-PCR-OUTR PCR. The outer PCR wasdiluted by 500,000 times and 1 μl of that was used as a template forinner PCR using primers HCV-PCR-INF and HCV-PCR-INR.

Template Preparation

The biotinylated PCR products were immobilized onto streptavidin-coatedsuper paramagnetic beads Dynabeads™ M280-Streptavidin (Dynal BiotechASA, Oslo, Norway). Single-stranded DNA was obtained by discarding thesupernatant after incubation of the immobilized PCR product in 0.10 MNaOH for 3 min. Five pmol of sequencing primers HCV-SEQF1, HCV-SEQF2,and HCV-SEQF3 were hybridized to the immobilized strand, as described inRonaghi et al., 1996, Analytical Biochemistry, 242, 84-89.

Primer Extension Reaction

The primed DNA templates were placed in a microtiter plate containing0.5 μg SSB (Amersham Pharmacia Biotech, USA), and Pyrosequencing™substrates and enzymes (www.pyrosequencing.com Pyrosequencing AB,Uppsala, Sweden) nucleotides were dispensed using fully automatedmicrotiter plate-based PSQ™ Pyrosequencing™ instrument. The sequencingprocedure was carried out by stepwise elongation of the primer-strandupon pre-specified addition of four different nucleotides. The templatewas hybridized with the three extension primers described above. Theprogress of sequencing was followed in real-time using Pyrosequencing™Tag software, (Pyrosequencing™ AB, Uppsala, Sweden) and subtyping wasperformed manually.

HCV positive blood sera from 89 different patients was collected and HCVRNA was extracted as described above. Subsequent to cDNA synthesis, PCRwas performed to amplify a 236-base long region from 5′ UR. One of theprimers in the PCR was biotinylated. After capture of the PCR productson magnetic beads and template preparation, sequencing-by-synthesis wasperformed.

Results

Principle of the HCV Typing Method.

The principle of the typing method described above is outlined inFIG. 1. In this model system, extension primers are hybridized to thetarget sample DNA, which is immobilized on magnetic beads.

The extension primers hybridise specifically to the conserved regionadjacent to the variable region. In this set of experiments, 3sequencing primers for HCV were used. The primers and their alignment tothe HCV genomes are shown in FIG. 2.

The signals resulting from the specific extension of each primer aredirectly correlated to the number of nucleotides incorporated. The‘fingerprint’ produced can therefore be used to identify the genotype ofthe individual, against reference fingerprints, which can betheoretically deduced from the sequences of the variable regions.References fingerprints calculated theoretically from the sequence ofthe variable regions are shown on FIG. 3. These can be used to type theresults shown on FIG. 4: FIG. 4 a is the fingerprint for HCV 1 a, FIG. 4b is the fingerprint for HCV 1 b, FIG. 4 c is the fingerprint for HCV 2a, FIG. 4 d is the fingerprint for HCV 2 b, FIG. 4 e is the fingerprintfor HCV 3 a, and FIG. 4 f is the fingerprint for HCV 3b. Therefore,using the method of the invention, it was possible to genotype HCVinfection. Of the 77 sera analyzed by the method of the invention. 350were infected with HCV 1 a, 29% with HCV 1 b, 21% with HCV 2 a, 4% withHCV 2 b, 1% with HCV 3 a and 10% with HCV 3 b. Of the 10 analysedsamples from Iran, the following results were obtained; 1 a, 1; 1 b, 3;2 a, 3; 3 a, 2 and 3 b; 1.

EXAMPLE 2

Typing of SNPs in the RAAS System

Templates and Primers

Genomic DNA was isolated according to standard methods, PCR temples wasgenerated with specific primers according to the table below. PCR PCRprimer 1 From ref. fragment 5′-biotin PCR primer 2 U.S. Pat. No. 6197505Eu3 GGA CCA GCT CTC CAC AGT GC GCC AGC ACG TCC CCA AT ACEe8R (PCR2) Eu4GAT TCC CCT CTC CCT GTA CCT GCC AGG AAG TTT GAT GTG AAC ACEe15R (PCR1)Eu6 CTC GCT CTG CTC CAG GTA C GCC TCC TTG GAC TGG TAG AT ACEe24F (PCR2)Eu8 CCA GGG CAG GGC TGA TA CAA ACG GCT GCT TCA GGT ANGe2f3F Eu10 CAT TTCTTG GTT TGT TCT TCT GA GTT TGT GCT TTC CAT TAT GAG TC AT1e5f3F

The following sequencing primers were used in the multiplex reactions:Eu3 Eu3s 5′-CCC CGA CGC AGG GAG AC-3′ A062RS 5′-CCC CGA CGC AGG GAG-3′A0943S 5′-CCC CGA CGC AGG G-3′ Eu4 Eu4s 5′-GAC CTA GAA CGG GCA GC-3′A097FS 5′-GTT CAG GAC CTA GAA-3′ Eu6 Eu6s 5′-CCT CGC TCC GCT CCA GGTA-3′ A091FS 5′-CTC GCT CTG CTC-3′ A063FS 5′-CTC GCT CTG CTC CAG GT-3′Eu8 A089RS (Eu8s) 5′-GCT GTG AAC ACG CCC AC-3′ A060FS 5′-GCT GCT GCT GCTCA-3′ Eu10 A088FS (Eu10s) 5′-AGA TCC CAA AAT TCA ACC CT-3′PCR Amplification

The target nucleic acid molecules were amplified by PCR, either bystandard PCR or by multiplex PCR.

Simplex PCR: A 50 μl PCR reaction was set up for each SNP-specificfragment and sample. All fragments were amplified with the AmpliTaq Goldkit (PE Biosystems) and 1.5 mM MgCl₂ according to the followingprotocol. (Table 1). TABLE 1 PCRmix 1× 100× 10 × PCRbuffer 5 500 MgCl₂(25 mM) 3 300 dNTP (2.5 mM) 2.5 250 DMSO 0 0 Primer a (10 μM) 1 100Primer b (10 μM) 1 100 TaqGold (5 units/μl) 0.3 30 H₂O 32.2 3220 Sum: 4545005 μl genomic DNA (2 ng/μl) was added to 45 μl PCR mix.PCR Cycling Conditions:

95° C. 5 min, 50×(95° C. 15s, 57° C. 30s, 72° C. 45s), 72° C. 5 min, 4°C.

Multiplex PCR using 4 amplification primers: A 50 μl PCR reaction wasset up using Eu4 and Eu8 SNP-specific fragments. All samples wereamplified with the HotStarTaq Master Mix Kit from Qiagen addingQ-solution and MgCl₂to a final concentration of 2.0 mM according to thefollowing protocol (Table 3). TABLE 3 Magnesium concentration 2.0 mMPCRmix 1× 100× 10 × PCRbuffer (15 mM MgCl₂) 5 500 MgCl₂ (25 mM) 1 100dNTP (2.5 mM) 2.5 250 Q-solution 10 1000 Primer 4a (10 μM) 2 200 Primer4b (10 μM) 2 200 Primer 8a (10 μM) 2 200 Primer 8b (10 μM) 2 200 TaqGold(5 units/μl) 0.25 25 H₂O 13.25 1325 Sum: 40 400010 μl genomic DNA (2 ng/μl) was added to 40 μl PCR mix.PCR Cycling Conditions:

95° C. 15 min, 35×(94° C. 30s, 55° C. 1 min, 72° C. 2 min), 72° C. 10min, 4° C.

Multiplex PCR using 6 amplification primers: A 50 μl PCR reaction wasset up using Eu3, Eu6 and Eu10 SNP-specific fragments. All samples wereamplified with the HotStarTaq Master Mix Kit from Qiagen addingQ-solution and MgCl₂ to a final concentration of 2.0 mM according to thefollowing protocol (Table 4). TABLE 4 Magnesium concentration 2.0 mMPCRmix 1× 100× 10 × PCRbuffer 5 500 MgCl₂ (25 mM) 1 100 dNTP (2.5 mM)2.5 250 Q-solution 10 1000 Primer 3a (10 μM) 2 200 Primer 3b (10 μM) 2200 Primer 6a (10 μM) 2 200 Primer 6b (10 μM) 2 200 Primer 10a (10 μM) 2200 Primer 10b (10 μM) 2 200 TaqGold (5 units/μl) 0.25 25 H₂O 10.25 1025Sum: 40 450010 μl genomic DNA (2 ng/μl) was added to 40 μl PCR mix.PCR Cycling Conditions:

95° C. 15 min, 35×(94° C. 30s, 59° C. 1 min, 72° C. 2 min), 72° C. 10min, 4° C.

Sample Preparation

25 μl of PCR product (multiplex PCR product or pooled standard PCRproduct) was immobilised by the addition of 10 μl Dynabeads™ (DynalBiotech ASA, supra) (10 μg/μl) together with 25 μl 2×BW buffer (10 mMTris-HCl pH 7.57, 2M NaCl, 1 mM EDTA and 0.1% Tween 20). 15 pmolsequencing primer was added in annealing buffer (20 mM Tris-Acetate pH7.51, 5 mM MgAc2) and the mixture incubated for 2 minutes at 80° C. Thesamples were then allowed to cool to room temperature. 2.2 μg SSB(Amersham Pharmacia Biotech, supra) may be added at this point, ifrequired.

Primer Extension

The primed DNA templates were placed in a microtiter plate containingPyrosequencing™ substrates and enzymes (PSQ96™ plate, Pyrosequencing AB,supra). Nucleotides were dispensed using fully automatedmicrotiter-plate based PSQ™ Pyrosequencing™ instrument. The sequencingprocedure was carried out by stepwise elongation of the primer-strandupon pre-specified addition of four different nucleotides. The templateswere hybridized with the extension primers mentioned above. The progressof sequencing was followed in real-time using Pyrosequencing™ software.

Results

Principle of the SNP Typing Method.

The principle of the typing method described above is outlined in figureseven. In this model system, extension primers are hybridized to thetarget sample DNA, which is immobilised on magnetic beads.

In this set of experiments 3 sequencing primers for the RAAS system wereused, either in isolation to show the ‘simplex patterns’ or incombination to show the multiplex patterns.

The signals resulting from the specific extension of each primer aredirectly correlated to the number of nucleotides incorporated. The‘fingerprint’ produced can therefore be used to identify the genotype ofthe individual, against reference fingerprints, which can betheoretically deduced from the sequences of the variable regions.Reference fingerprints calculated theoretically from the sequence of theSNPs are shown on FIG. 8. 8 a is the theoretical output for SNP Eu4 (ACEG2215A), 8 b is the theoretical output for SNP Eu8 (ATG C521T) and 8 cis the theoretical output for the simultaneous analysis of SNPs Eu4 andEu8, the polymorphic positions are framed. 8 d is the theoretical outputfor SNP Eu10 (ATP T573C), 8 e is the theoretical output for SNP Eu6 (ACET3409C), 8 f is the theoretical output for Eu3 (ACE T1237C) and 8 g isthe theoretical output for the simultaneous analysis of SNPs, Eu10, Eu6and Eu3. SNPs Eu4, Eu10 and Eu6 are shown as heterozygotes, SNPs Eu8 ashomozygote G and SNP Eu3 as homozygote A. These “reference” patterns canbe used to type the results shown in FIG. 9: 9 a is the sequencing datafor SNP Eu4 (A/G), 9 b is the sequencing data for SNP Eu8 (G/G) and 9 cis the multiplex sequencing data for the combination of SNP Eu4 (A/G)and SNP Eu8 (G/G), which correlates to theoretical output 8 c, theframes indicating SNP positions. 9 d, 9 e and 9 f are the sequencingdata plots for SNP Eu10 (C/T), Eu6 (C/T) and Eu3 (A/A), respectively,and 9 g is the multiplex sequencing data for the combination of these 3SNPs, the polymorphic positions are boxed. Pattern 9 g correlates toFIG. 8 g.

EXAMPLE 3

Triplex genotyping on 4 SNPs in the RAAS System—Eu7 Eu8 (containing 2SNPs), and Eu11.

Templates and Primers PCR PCR primer 1 From ref. fragment 5′-biotin PCRprimer 2 U.S. Pat. No. 6197505 Eu7 TGA TGT AAC CCT CCT CTC CA CGG CTTACC TTC TGC TGT ANPf4F AGT A Eu8 CCA GGG CAG GGC TGA TA CAA ACG GCT GCTTCA GGT ANGe2f3F Eu11 TTT CTC CTT CAA TTC TGA GCC CCT CAG ATA ATG TAAAT1-spec.1 AAA GTA GC

Sequencing Primers Eu7 Eu7s 5′-ACG GCA GCT TCT TCC CC-3′ Eu8 A089RS(Eu8s) 5′-GCT GTG AAC ACG CCC AC-3′ A060FS 5′-GCT GCT GCT GCT CA-3′ Eu11Eu11s 5′-GCA GCA CTT CAC TAC CAA AT-3′

PCR Amplification, sample preparation and primer extension reactionswere performed as described in Example 2, with the exception of Eu11,which was amplified according to the protocol in Table 2. TABLE 2 PCRmix1× 100× 10 × PCRbuffer 5 500 MgCl₂ (25 mM) 3 300 dNTP (2.5 mM) 2.5 250DMSO 0 0 Primer 11a (10 μM) 2 200 Primer 11b (10 μM) 2 200 TagGold (5units/μl) 0.3 30 H₂O 30.2 3020 Sum: 45 45005 μl genomic DNA (2 ng/μl) was added to 45 μl PCR mix.PCR Cycling Conditions for Eu11:

95° C. 5 min, 50×(95° C. 315s, 52° C. 30s, 72° C. 45s), 72° C. 5 min, 4°C.

Results

In this set of experiments 3 sequencing (“extension”) primers for theRAAS system were used, and the signals resulting from the specificextension of each primer can be directly correlated to the number ofnucleotides incorporated. Theoretical reference patterns are shown inFIG. 10, which can be used to determine the genotype shown in FIG. 11.10 a, 10 b and 10 c are the theoretical outputs obtained for SNPs Eu8(G/G), Eu7 (C/C and T/T) and Eu11 (A/G), with the theoretical multiplexoutput shown on FIG. 10 d. This correlates to the actual resultsobtained shown in FIG. 11. The polymorphic positions are boxed, and thegenotype of this individual is Eu8 G/G, Eu7 C/C and T/T and Eu11 A/G.The pyrogram exhibits some nucleotide background incorporation which canbe reduced as discussed previously (e.g. add SSB after primerannealing).

EXAMPLE 4

SNP typing in Human Coagulation Factor V, Prothrombin and Plasminogenactivator inhibitor.

Introduction

Thrombosis is a complex (multifactorial) trait. The genes involved aretypically susceptibility genes, where the differences are not pointmutations but particular forms (alleles) of polymorphisms. The disorderresults from the presence of an increased frequency of specific allelesin unfavorable combinations.

During the last ten to fifteen years, mutation or variation in severalgenes has been found to be associated with venous thrombosis. Thisincludes genes such as factor V (FV), prothrombin (FII) and plasminogenactivator inhibitor (PAI1).

Coagulation Factor V (FV) and Prothrombin (FII) are both essentialcomponents in the human coagulation cascade, which ultimately results inthe stemming of blood loss. Prothrombin is proteolytically cleaved inthe first step of this cascade converting into the clotting enzymethrombin. Coagulation factor V serves as a cofactor for the coagulationfactor X-catalyzed activation of prothrombin to thrombin. Pointmutations in these genes may cause impairments in processes ofthrombosis and hemostasis. One such is venous thrombosis, predominantlyafflicting people of European origin. The mutations, Factor V Leiden(FV:G1691A) and the G20210-A prothrombin variant (FII:G20210A), are thetwo single most important genetic risk factors for developing venousthrombosis. This European predisposition has been explained to someextent by the characterization by these two variants. In addition tothese two established risk factors for venous thrombosis, the role ofother genetic variations is still under investigation (Martnelli et al.,1998; De Stefano et al., 1999; Rees et al., 1999; Hessner et al., 1999).

Several prospective studies have documented that the fibrinolyticcapacity is an important determinant of the risk of thrombosis. Manystudies have convincingly shown that survivors of myocardial infarctionhave impaired fibrinolytic activity because of increased concentrationsof plasma plasminogen activator inhibitor-1 (PAI-1). A single guanosineinsertion/deletion polymorphism in the promoter region of the PAI1 gene,commonly called 4G/5G, has been shown to be associated with plasma PAI-1activity (Dawson et al, 1993; Eriksson et al., 1995).

Primers

Three sets of PCR primers were designed. The fragment spanning over exon10 and intron 10 of human coagulation factor V was 162 bp long, theprothrombin fragment spanning over exon 14 and intron 14 was 211 bp andthe fragment in the promotor region of the PAI1 gene was 152 bp. Oneprimer in each set was biotinylated in order to allow subsequentimmobilization to magnetic beads/sepharose beads. In addition, threesequencing primers were designed to hybridize in close proximity to thefactor V Leiden SNP, the G20210A prothrombin variant and the 4G/5Gdeletion of PAI1 see FIG. 12.

PCR Primers: Prothrombin A001FPB Biotin-5′-CCT GAA GAA GTG GAT ACA GAAGG-3′ A008RP 5′-CAG TAG TAT TAC TGG CTC TTC CTG A-3′ Factor V PSO905′-GGG CTA ATA GGA CTA CTT CTA ATC-3′ PSO91B Biotin-5′-TCT CTT GAA GGAAAT GCC CCA TTA-3′ PAI1 PSO112FPB Biotin-5′-CCC ACC CAG CAC ACC TC-3′PSO113RP 5′-GAC TCT TGG TCT TTC CCT CAT C-3′

Sequencing Primers: Prothrombin A009SR 5′-ACT GGG AGC ATT GAG-3′ FactorV PSO83 5′-AGC AGA TCC CTG GAC-3′ PAI1 A114SR 5′-CAC GGC TGA CTC CCC-3′PCR Amplification

A 50 μl PCR reaction was set up using HotStarTaq Master Mix Kit fromQiaGen according to the following protocol TABLE 5 Magnesiumconcentration 2.0 mM PCRmix 1× 100× 10 × PCRbuffer (15 mM MgCl₂) 5 500MgCl₂ (25 mM) 4 400 dNTP (2.5 mM) 2.5 250 A001FPB (10 mM) 1 100 A008RP(10 mM) 1 100 PSO90 (10 mM) 1 100 PSO91B (10 mM) 1 100 PSO112FPB (10 mM)1 100 PSO113RP (10 mM) 1 100 HotStarTaq (5 units/ml) 0.2 20 H₂O 29.32930 Sum: 45 45005 μl genomic DNA (2 ng/μl) was added to 45 μl PCR mix.PCR Cycling Conditions:

95° C. 5 min, 50×(95° C. 30s, 67° C. 45s, 72° C. 60s), 72° C. 5 min, 4°C.

Sample Preparation and Primer Extension

Were performed as described in Example 2.

Results The theoretical output obtained by typing each SNP or deletionindividually are shown as FIGS. 13 a, 13 b and 13 c, representing PAI1genotype for 4G/5G deletion (C/C), SNP G20210A prothrombin (C/T) and SNPG1691A Factor V Leiden (A/G), respectively. The theoretical multiplexingoutput for the multiplex assay of these 3 SNPs is shown as FIG. 13 d,with the deletion or SNP position shown. FIGS. 13 e to 13 j representthe theoretical output expected for 6 genotypes upon which real data wasthen collected, see FIG. 14. The pyrograms shown in FIG. 14 are 6possible genotypes that can be present in the human population in thesegenes. 14 a is the results from the genotype PAI1 C/C, prothrombin C/Cand factor V G/G, 14 b is the genotype PAI1 del/del, Prothrombin C/C andfactor V A/A, 14 c is the genotype PAI1 del/C, Prothrombin T/T andFactor V G/G, 14 d is the genotype PAI C/C, prothrombin T/C and Factor VG/G, 14 e is the genotype PAI1 C/del, Prothrombin C/C, Factor V G/G and14 f is the genotype PAI1 C/C, prothrombin C/C and Factor. V A/G. FIG.14 a corresponds to 13 e, 14 b to 13 f, 14 c to 13 g, 14 d to 13 h, 14 eto 13 i and 14 f to 13 j.

EXAMPLE 5

CYP2D6 SNP Analysis

Introduction

The CYP2D6 gene is a member of the cytochrome P450 gene superfamily,which in total consists of nine gene families. Four of these genefamilies are responsible for the metabolism and elimination of mostforeign chemicals that enters the body via ingestion. The human CYP2Dlocus is mapped to chromosome 22q13.1 (Gough et. al, 1993). The CYP2D6gene encodes for an enzyme, debrisoquine 4-hydroxylase, which isinvolved in the metabolism of more than 40 drugs, among themneuroleptics, antidepressants, anthiarrhytmics, b-blockers and opioids.The enzyme is characterised by extreme variability in activity(interindividual and interethnic). The CYP2D6 genotype and catalyticfunction are closely coupled, and genotyping could be an important toolfor determining drug doses for individuals. More than 50 alleles havebeen identified, of which many encodes for a non-functional enzyme Thealleles are defined by a number of variations; SNPs, insertion ordeletions of single base pairs, deletion of the complete gene, andduplications of the gene. The sequences analysed in this example are asfollows: G1846T: GCCAACCACTCC G/T GT G1934A: G/A GACGCCCCTTCG T1795del:GCAG (T) GGGTGACCG G1749C: G/C CTCCACCTTGCGPrimers

Table 6. PCR primers and sequencing primers in the multiplex method. Theprimers are named F for a forward direction and R for a reverseddirection. P represents a PCR primer, and S a sequencing primer and Bmeans biotin labelled in the 5′ end. Sequence Frag- to be ment PrimersPrimer sequence identified 61118 A061RPB B-CCTCGGTCTCTCGCTCCGC A118FPGAGCAGAGGCGCTTCTCCGT A143FS CCTTCGCCAACCAC TCCG/TGT A182FSCAAGAAGTCGCTGGAG CAG (T) GGGTG A183FS GCATCTCCCACCCCC AG/ AGACGCCCCTTTC2162 A021RPB B-ACTGTTTCCCAGATGGGCTC A062FP GACCCCGTTCTGTCTGGTGT A145FSTTCAATGATGAGAACC TGC/TG A146FS CCTGCTCATGATCCT ACA/CTCCGG A147FSTGAGCTGCTAACTGA GCAC (A) GGFIG. 15 shows the localisation of the primers in the CYP2D6 nucleic acidfragments for the multiplex method: fragments 2162 and 61118.PCR Amplification

A nested PCR amplification was performed. For both the first and thenested 50 μl PCR reaction HotStarTaq Master Mix Kit from QiaGen was usedand was set up according to the following protocol (Table 7). TABLE 7Magnesium concentration 1.5 mM PCRmix 1× 100× 10 × PCRbuffer (15 mMMgCl₂) 5 500 MgCl₂ (25 mM) 0 0 dNTP (2.5 mM) 4 400 Primer 1 (10 mM) 1100 Primer 2 (10 mM) 1 100 HotStarTaq (5 units/μl) 0.5 50 H₂O 37.5 3750Sum: 49 4900PCR 1.

1 μl genomic DNA (10 ng/μl) was added to 49 μl PCR mix.

PCR Cycling Conditions:

PCR method, primary PCR (fragment 4142) 95° C. 15 min, 25×(95° C. 45s,66° C. 45s, 72° C. 60s), 72° C. 5 min, 4° C.

PCR Method, Secondary PCR

95° C. 5 min, 20×(95° C. 45s, T_(A) 45s, 72° C. 45s), 72° C. 5 min, 4°C.

T_(A), fragment 61118 was 61° C.

2162 was 63° C.

Sample Preparation

Took place as described in example 2. SSB was added to theprimer/template mix after hybridisation. 0.55 μg SSB was added forfragment 2162 and 2.2 μg for fragment 61118. The amounts of sequencingprimers were for fragment 2162: 15 pmoles of each, and for fragment61118: 5 pmoles of primers 182 and 183, and 70 pmoles of primer 143.

Primer Extension

Was performed as described for example 2.

Results

Two theoretical output for fragment 61118 in a multiplex analysis areshown as FIGS. 16 a and 16 b. 16 a showns the genotype G₁₉₃₄A (A/G),G₁₇₄₉C (C/G), T₁₇₉₅del (no deletion) and G₁₈₄₆T (T/G) and FIG. 16 bdiffers in that T₁₇₉₅del shows the deletion of the T residue.

FIG. 17 shows the actual results from genotype established from thepyrogram is G₁₉₃₄A (G/G), G₁₇₄₉C (G/G), T₁₇₉₅del (no deletion ∴T/T) andG₁₈₄₆T (G/G). This is a different genotype to those shown in FIG. 16.

This demonstrates that it is possible to type multiple SNPs anddeletions on one fragment of nucleic acid using multiple extensionprimers.

EXAMPLE 6

Serum Samples

72 sera from HCV-positive Veterans were obtained from Stanford Veteranhospital. Five HCV-positive sera were obtained from Iran.

Synthesis and Purification of Oligonucleotides

The oligonucleotides HCV-PCR-OUTF (5′-CCCTGTGAGGAACTWCTGTCTTCACGC),HCV-PCR-OUTR (5′-GCTCATGRTGCACGGTCTACGAGACCT), HCV-PCR-INF(5′-TCTAGCCATGGCGTTAGTAYGAGTGT), BHCV-PCR-INR(5′-Biotin-CACTCGCAAGCACCCTATCAGGCAGT), HCV-SEQF1(5′-GGAACCGGTGAGTACACCGGAAT), HCV-SEQF2 (5′-GACYGGGTCCTTTCTTGGA),HCV-SEQF3 (5′-ATTTGGGCGTGCCCCCGC), were all synthesized and HPLCpurified by MWG Biotech (High points, N.C., USA).

RNA Extraction, cDNA Synthesis and Amplification

RNA was extracted from 50 μl of serum. cDNA was synthesized using AMVreverse transcriptase on HCV cDNA obtained from different patients usingBHCV-PCR-INR and HCV-PCR-INF to generate a 270 base long product.

The biotinylated PCR products were immobilized onto streptavidin-coatedsuper paramagnetic beads Dynabeads™ M280-Streptavidin (Dynal A. S.,Oslo, Norway). Single-stranded DNA was obtained by removing thesupernatant after incubation of the immobilized PCR product in 0.10 MNaOH for 3 min. Five pmol of sequencing primers HCV-SEQF1, HCV-SEQF2,and HCV-SEQF3 were hybridized to the immobilized strand.

Primer Extension Reaction

The primed DNA template were placed in a microtiter plate containing 0.5μg SSB (Amersham Pharmacia Biotech, USA), and Pyrosequencing™ substrates(www.pyrosequencing.com Pyrosequencing AB, Uppsala, Sweden) and enzymeswere dispensed using fully automated microtiter plate-based PSQ™Pyrosequencing™ instrument. The sequencing procedure was carried out bystepwise elongation of the primer-strand upon pre-specified addition offour different nucleotides. The template was hybridized with the threeextension primers described above. The progress of sequencing wasfollowed in real-time using Pyrosequencing™ SNP software,(Pyrosequencing™ AB, Uppsala, Sweden) and subtyping was performedmanually.

Results

Principle of the Typing Method.

The principle of the typing method described above is outlined inFIG. 1. In this model system, extension primers are hybridized to thetarget sample DNA, which is immobilized on magnetic beads.

The extension primers hybridise specifically to the conserved regionadjacent to the variable region.

The signals resulting from the specific extension of each primer aredirectly correlated to the number of nucleotides incorporated. The‘fingerprint’ produced can therefore be used to identify the genotype ofthe individual, against reference fingerprints, which can betheoretically deduced from the sequences of the variable regions.References fingerprints calculated theoretically from the sequence ofthe variable regions are shown on FIG. 3. These can be used to type theresults shown on FIG. 4: FIG. 4 a is the fingerprint for HCV 1 a, FIG. 4b is the fingerprint for HCV 1 b, FIG. 4 c is the fingerprint for HCV 2a, FIG. 4 d is the fingerprint for HCV 2 b, FIG. 4 e is the fingerprintfor HCV 3 a, and FIG. 4 f is the fingerprint for HCV 3 b. Therefore,using the method of the invention, it was possible to genotype HCVinfection. Of the 77 sera analyzed by the method of the invention. 35%were infected with HCV 1 a, 29% with HCV 1 b, 21% with HCV 2 a, 4% withHCV 2 b, 10% with HCV 3 a and 1% with HCV 3 b.

1. A method of typing one or more nucleic acid molecules, said methodcomprising: simultaneously hybridizing two or more extension primers tosaid nucleic acid molecule or molecules and performing primer extensionreactions therefrom, each primer binding at a different predeterminedsite in said nucleic acid molecule or molecules, and determining thepattern of nucleotide incorporation by sequencing-by-synthesis to obtaina test pattern for said nucleic acid molecule or molecules which isoptionally compared with one or more reference patterns to type the saidnucleic acid molecule or molecules.
 2. A method as claimed in claim 1wherein the nucleic acid contains two or more variable sites.
 3. Amethod for obtaining typing information about a plurality of variablesites within target nucleic acid, comprising simultaneously hybridizingtwo or more extension primers to said nucleic acid and performing primerextension reactions therefrom, each primer binding at a differentpredetermined site in said target nucleic acid, the pattern ofnucleotide incorporation determined from said primer extension reactionsby sequencing-by-synthesis providing the typing information about saidvariable sites.
 4. (Cancelled).
 5. A method as claimed in claim 1wherein nucleotides are added to the reaction mix sequentially in apredetermined order.
 6. A method as claimed in claim 1 whereinnucleotide incorporation is determined quantitatively.
 7. A method asclaimed in claim 1 wherein if nucleotide incorporation takes place atone variable site, there is no nucleotide incorporation at the othervariable site(s).
 8. A method as claimed in claim 1 wherein a firstextension primer binds closer to its variable site than a second primerdoes to its variable site.
 9. A method as claimed in claim 8 wherein thesecond primer is 10-20 nucleotides further away from its variable sitethan is said first primer.
 10. A method as claimed in claim 1 whereinsingle-stranded binding protein is added to the reaction mix after theprimers are annealed to the nucleic acid template.
 11. A method asclaimed in claim 1 wherein the primer extension reactions occursimultaneously.
 12. A method as claimed in claim 1 wherein 3 or morevariable sites are typed.
 13. A method as claimed in claim 1 wherein 3or more primer extension reactions are performed.
 14. A method ofdiagnosis of pathological conditions characterised by the presence ofspecific nucleic acid molecule or molecules, comprising simultaneouslyhybridizing two or more extension primers to said nucleic acid moleculeor molecules, and performing primer extension reactions therefrom, eachprimer binding at a different predetermined site in said nucleic acidmolecule or molecules, the pattern of nucleotide incorporation,determined from said primer extension reactions bysequencing-by-synthesis, allowing diagnosis of said pathologicalconditions.
 15. A kit for use in a method of typing nucleic acid whichcomprises: optionally one or more primers for in vitro amplification;two or more primers for primer extension reactions each primer bindingat a different predetermined site in a nucleic acid molecule;nucleotides for amplification and/or for the primer extension reaction;optionally a polymerase enzyme for the amplification and/or primerextension reaction; and optionally means for detecting primer extension.